In Vivo Competition between Plastocyanin and a Copper-Dependent Regulator of the Chlamydomonas reinhardtii Cytochrome c(6) Gene

Abstract

The Chlamydomonas reinhardtii gene encoding cytochrome c(6) (Cyt c(6)) is transcriptionally repressed by cupric ions. In quantitating the level of expression of this gene as a function of cupric ions available per cell, we find that transformed Chlamydomonas reinhardtii cells that accumulate high levels of plastocyanin (a type I copper protein) have a higher sensory threshold for copper-dependent repression of the Cyt c(6) gene than do untransformed, otherwise isogenic, cells that are plastocyanin-deficient. Also, in wild-type cells, the extent to which the gene is expressed at any given ratio of copper/cell is exactly correlated with the predicted deficiency (at this level of copper) in the organism's capacity to synthesize holoplastocyanin. These results support a simple model in which the sensory threshold for transcriptional repression of the Cyt c(6) gene is determined by direct competition for intracellular copper ions between a copper-binding regulator of this gene and plastocyanin. Thus, the organism is able to maintain a constant amount of Cyt c(6) plus plastocyanin per Photosystem I. With the use of in vitro-generated Cyt c(6)-encoding transcripts as a standard for the quantitation of cellular Cyt c(6) mRNA levels, we estimate that whereas copper-deficient wild-type cells maintain approximately 1 x 10(2) to 4 x 10(2) Cyt c(6)-specific transcripts per cell, copper-supplemented cells contain, on average, less than one Cyt c(6)-encoding mRNA. Thus, repression of the Cyt c(6) gene by copper ions is essentially 100%, making it unlikely that Cyt c(6) has any essential metabolic function in copper-supplemented cells. We find also that the steady-state levels of several transcripts, including those for Cyt c(6), are influenced by cell density, so that cells harvested at low density contain several-fold as many copies of a particular message as cells harvested near stationary phase.